Project Summary The waterborne pathogen, Giardia lamblia, is a major cause of parasite-induced diarrheal diseases worldwide, including the U.S. Giardia has a biphasic life cycle?i.e., replicative trophozoites and relatively dormant but viable cysts. While cysts are responsible for the transmission of Giardia through sewage-contaminated water, trophozoites colonize the upper portion of the human small intestine and cause the infection (called ?giardiasis?), which can be symptomatic or non-symptomatic. The symptoms of giardiasis include diarrhea, irritable bowel syndrome (IBS), indigestion and gastro-intestinal discomfort. Children in low-resource settings are especially vulnerable to the infection and often suffer from malnutrition and age-specific development. Clinical resistance has been reported for all prescribed anti-giardial agents, including metronidazole with reports of treatment failure rates increasing from 15% in 2008 to 40% in 2013. We recently reported that oseltamivir was active against Giardia in culture. This led to our discovery that the active metabolite of oseltamivir (oseltamivir-free acid or OFA) is a potent inhibitor of trophozoite growth and cyst formation in laboratory culture. Because oseltamivir inhibits neuraminidases, our further investigation led to the discovery of a previously unreported giardial sialidase (gSial). This application is focused on exploitation of the abovementioned discoveries. Specifically, we aim to examine if this novel non-metronidazole class of anti-giardial agents has clinical potential for treatment of giardiasis, as well as to elucidate their mechanism of action. These anti-giardia agents are likely to have a different mode of action from current agents (inhibition of giardial sialidase) and therefore should be active against strains of Giardia resistant to current therapeutics. In Specific Aim-1, we will synthesize and test several OFA analogues on giardial growth and encystation in vitro (in culture) and in vivo (in mice). In Specific Aim-2, we seek to characterize gSial (a presumed target of OFAs in Giardia) by biochemical and molecular methodologies. The interactions between OFAs and gSial will also be investigated. Thus, the successful completion of this project?s aims should lead to the discovery of a novel lead for further development against giardiasis and validation of giardia sialidase as a novel drug target for giardiasis. Much is known about oseltamivir and its active metabolite and this should facilitate repositioning of this class of drugs for giardiasis.